The cell membrane is one of the major barriers for an application of therapeutically interesting bio-macromolecules like nucleic acids. In the present study we exploited a CPP-approach for the delivery of siRNA as a general example for an oligonucleotide cargo using the peptide MPGα, a derivative of the original MPG peptide described by Morris et al
). MPGα differs from MPG by six amino acids in the hydrophobic part (57
). These changes result in an alteration of the overall structure of the peptide towards a higher tendency of adopting a helical conformation (57
). The peptide forms stable non-covalent complexes with nucleic acids. Besides the mechanism of cellular uptake of MPGα/siRNA complexes our main focus was a comparative parallel analysis of uptake versus functional effects of the nucleic acid cargo. For this purpose, we used a liquid hybridisation protocol in combination with a simple luciferase reporter system. For comparison, the commercially available cationic lipid LF2000 was included into the study.
As shown in , MPGα is capable of translocating siRNA into mammalian cells leading to a down-regulation of the target protein luciferase. The observed effect was highly specific for siR206 and maximal inhibition was achieved with a charge ratio of 15:1, i.e. positive (peptide) over negative (siRNA) charges (Supplementary Figure 1). Additionally, we tested MPGα-mediated delivery of siRNA directed to two other targets, namely ICAM-1 and lamin A/C applying published protocols (58
). In both cases we observed a substantial RNAi effect of the active siRNA as compared to a control siRNA (data not shown). These data clearly indicate a specific siRNA-mediated inhibition of target protein expression rather than an unspecific effect, which might be caused by the transfection procedure. Comparing the peptide with LF2000, the maximal achievable levels of siRNA-mediated target protein down-regulation are roughly the same. However, an exact determination of the IC50
values of both carrier/cargo complexes revealed an ~30-fold lower efficiency of MPGα compared to LF2000. To investigate if this phenomenon was caused by different levels of uptake or insufficient bio-availability of the siRNA molecules, we quantified the intracellular amount of cargo. From a number of diverse procedures described in the literature (65
) we adapted a liquid hybridisation protocol described by Overhoff et al
). This assay does not need any modification of the siRNA and exclusively detects intact molecules. Thus, artefacts due to detached fluorescence labels or degraded cargos for example are precluded. Overall, the liquid hybridisation protocol is a fast, easy to handle and highly reproducible procedure that can be carried out in any laboratory without the need of expensive equipment.
Though, before we could perform quantitative uptake experiments, we had to establish a washing procedure in order to remove complexes bound to the surface of the cells. Such extracellularly attached carrier/cargo complexes are an important source of overestimation of cargo internalised (35
). As opposed to the commonly used trypsin treatment, a heparin wash was much more effective in our hands (Supplementary Figure 2A and B). Similar results were reported by Kaplan et al
) for the cellular uptake of Tat peptide in the absence of cargo. The negatively charged heparin molecules, with a molecular weight of 4–6 kDa, are much smaller than trypsin and thus might be able to infiltrate the extracellular matrix more effectively and both detach and, more importantly, dissociate MPGα/siRNA complexes bound there. In contrast to this, trypsin considerably increased the apparent uptake of the complexes (Supplementary Figure 2B). This might be due to a membrane destabilising effect of this enzyme which has been shown to enhance the uptake of small molecules or oligonucleotides (70
). For several CPPs without a cargo it has previously been shown that the cellular penetration is inhibited by certain GAGs suggesting that uptake is mediated by GAG receptors on the cell surface (30
). To what extent these receptors are equally involved in the uptake of MPGα/siRNA complexes cannot be answered with certainty as heparin has a very strong destabilising effect on the peptide/nucleic acid complexes (A.T., unpublished data).
With a reliable washing procedure to remove extracellular complexes in place we were able to accurately quantify the intracellular amounts of cargo after carrier-mediated delivery ( and ). The amount of siRNA internalised, increased as expected with the incubation time of transfection showing a maximum at ~4 h (Supplementary Figure 3B and C). Similar results were observed with a RNA aptamer [(72
), S.L., unpublished data]. Accordingly, unless otherwise indicated, all transfections were performed for a period of 4 h before cells were either subjected to quantification (after an additional 1 h heparin wash) or further incubated in the absence of carrier/cargo complexes for later analyses. After 24 h approximately half of the maximal amount of siRNA internalised was still detectable. All in all <5% of the siRNA present in the initial transfection mixture was internalised into the cells regardless of the delivery agent. After 4 h, twice as much siRNA was detected inside the cells after transfection with LF2000 when compared to MPGα-mediated delivery. Surprisingly, 24 h after transfection this difference rose to a factor of 4–6. In vitro
experiments, on the other hand, show that siRNA complexed with the carrier peptide is strongly protected from degradation (data not shown). The observed uptake of siRNA was linear over a tested range of 0.1–100 nM of siRNA. We deliberately did not apply concentrations >100 nM siRNA to examine if the process would eventually be saturable, since too high concentrations of siRNA lead to off-target effects (73
). Taken together, the difference in uptake between the peptide and the cationic lipid was much smaller than anticipated from the above described IC50
values with a factor of about 2–6 in favor of LF2000. These findings clearly indicate that uptake per se
was not the limiting factor of the peptide approach. Combining both techniques, the analysis of uptake and RNAi effect, it became apparent that for a half maximal inhibition of reporter gene activity, ~10
000 siRNA molecules were necessary in case of MPGα-mediated delivery, whereas in case of LF2000 only ~300 molecules were required. Thus, the amount of bio-available siRNA molecules inside the cells was about 30-fold lower for peptide-mediated delivery as compared to cationic lipid-mediated delivery. To the best of our knowledge, this is the first time the number of siRNA molecules per cell has been determined for half maximal inhibition of the target for peptide- versus cationic lipid-mediated delivery. At this point the question arose, what the cause of this observed discrepancy was. An obvious reason for these findings would be a difference in the mechanism of uptake. As outlined above, the mechanisms underlying the cellular translocation of CPPs are poorly understood and remain hotly debated. Nonetheless, there is considerable evidence that endocytosis is a major route of the internalisation of many CPPs. In an attempt to address this question we performed fluorescence microscopy studies and analysed the influence of specific inhibitors/effectors of different endocytotic pathways on the delivery of siRNA and the inhibition of reporter gene activity simultaneously.
A first clear hint for an endocytotic pathway involved in the uptake of MPGα/siRNA complexes arose from microscopy studies (). Consistent with the assumption of an endocytotic pathway, a typical vesicular distribution pattern in the cytoplasm could be observed by fluorescence microscopy. Moreover, the siRNA internalised was observed to partially co-localise with endosomal/lysosomal compartments (data not shown). The results were very similar for LF2000-mediated transfection though here a slight diffuse distribution of fluorescence throughout the cytoplasm was recognisable but hard to document. To avoid any artefacts due to cell fixation, all fluorescence microscopy experiments were performed with living cells. The heparin wash described above proved to be essential as images of cells not treated with heparin showed large aggregates on the cell surface impairing the view on complexes internalised (as clearly seen in Supplementary Figure 2A). Addition of trypan blue to quench external fluorescence (32
) was only suitable for smaller complexes but larger ones remained visible. Carboxyfluorescein in the medium turned out to be very useful to distinguish between the inside and the outside of the cell and was used to clearly discriminate between intra- and extracellular complexes. Although MPGα harbors an NLS sequence, no nuclear localisation could be observed nor was there any change in the apparent RNAi effect when the NLS sequence was mutated (data not shown). On the other hand, it cannot be ruled out that the amount of cargo localised in the nucleus was below the detection limit. According to a recent study of Berezhna et al
) the intracellular localisation of siRNA is suggested to depend on the localisation of the target. Nonetheless, applying a fluorescently labeled siRNA against the 7SK RNA, which was shown to strictly localise in the nucleus (77
), we did not observe any fluorescence there (data not shown).
Lowering of the temperature markedly reduces flexibility and fluidity of the plasma membrane, thereby also slowing membrane traffic (81
) and blocking all endocytotic processes (82
). Consistent with this concept, we could neither detect MPGα-mediated siRNA delivery at 4°C as shown by liquid hybridisation analyses as well as by fluorescence microscopy (Supplementary Figure 3), nor did we observe any RNAi effect (). In accordance with our findings, other authors also suggested an energy-dependent uptake process for CPP-mediated delivery based on the observation that uptake was strongly decreased at low temperatures (31
). In order to address this issue in more detail, we performed studies on MPGα with different modulators of endocytosis (). First we analysed the effect of wortmannin, a compound, which is supposed to affect clathrin-dependent endocytosis (85
) and macropinocytosis (86
). Wortmannin led to a strong reduction in the uptake of MPGα/siRNA complexes. Like with sucrose, which also blocks clathrin-dependent endocytosis unselectively, the RNAi effect was reduced significantly. On the other hand, no clear-cut influence on uptake of siRNA could be seen applying cytochalasin B, which is supposed to affect the assembly of actin microfilaments (87
), whereas the RNAi effect was strongly diminished in the presence of this compound. Hence, we could not distinguish, whether this was due to a blockage of macropinocytosis or what appears more realistic was caused by a block of intracellular vesicle transport. In the presence of nystatin, which is supposed to inhibit caveolin-dependent lipid-raft-mediated endocytosis (89
), the RNAi effect was clearly reduced and could neither be rescued by combining nystatin with okadaic acid, chloroquine nor filipin complex (). Pre-incubation of the cells with nystatin led to an increase in uptake during the first 4 h, which was even more pronounced when the quantification was performed after 24 h (). An additional effect of nystatin is supposed to be an increase in cell membrane permeability (89
). This property of nystatin could account for the high amount of siRNA taken up. Together with the reduced RNAi effect observed, this would mean that nucleic acids internalised this way, are not available for the RNAi machinery. Then again, taking the results of experiments into account in which different inhibitors were combined to counteract nystatin, it could be concluded that nystatin itself had a severe negative effect on the RNAi machinery. Filipin complex like nystatin shows sterol-binding properties (91
). However, in contrast to nystatin almost no change in siRNA uptake was detected after MPGα-mediated delivery, whereas the RNAi effect was slightly increased. This may be due to a selective inhibition of caveolin-mediated endocytosis, which in turn might shift the balance towards clathrin-mediated endocytosis. This interpretation would be consistent with our idea that clathrin-mediated endocytosis yields more bio-available cargo and is further supported by the observation that the RNAi effect is increased even more by combining filipin complex with chloroquine (). Okadaic acid, a specific phosphatase inhibitor, is supposed to stimulate mobility and internalization of caveolae (94
). In the presence of okadaic acid no RNAi effect could be detected. Uptake of siRNA, on the other hand, was not affected. This could be interpreted as an increase of caveolin-dependent endocytosis accompanied by a reduction of clathrin-dependent uptake leading to an unchanged net amount of intracellular siRNA. Caveolae direct most of their cargo via caveosomes to the Golgi apparatus where they are probably beyond reach of the RNAi machinery. In addition, combining filipin complex and okadaic acid resulted in levels of luciferase activity equal to those measured with siRNA alone. Here, induction of caveolin-mediated endocytosis by okadaic acid was possibly compensated by the inhibitory effect of filipin complex. Chloroquine, a weak base, is supposed to prevent lysosomal degradation by inhibiting the acidification of endosomes (97
). This eventually leads to the disruption of a large portion of the endosomes which then release their content into the cytoplasm (99
). In our hands, chloroquine caused a slightly enhanced RNAi effect leading to the conclusion that an increased number of siRNA molecules have become bio-available by endosomal release.
Equivalent experiments with LF2000 as transfection reagent yielded a slightly different pattern, for example regarding uptake in the presence of nystatin or chloroquine, nevertheless strongly indicating uptake via endocytosis (Supplementary Figure 4). This observation is consistent with the findings of others concerning the uptake mechanism of several cationic lipids (100
Overall, our data, especially those addressing the temperature-dependency of the uptake, strongly indicate that both MPGα and LF2000/siRNA complexes are taken up by endocytotic processes. The differences between transfections with MPGα and LF2000 concerning the IC50
values of siRNA could be explained by the property of LF2000 to promote endosomal escape at least partially, which is consistent with the microscopic observations described above. The somewhat controversial results concerning different inhibitors of endocytosis described above support the notion that not one single pathway is responsible for the uptake of the complexes but rather several pathways are involved. This may be due to a relatively unspecific interaction of the complexes with numerous negatively charged molecules on the cell surface. As a result, various pathways of endocytosis might be triggered. Accordingly, individual inhibitors used in this study might show just moderate effects by shifting the balance in favour of a particular endocytotic pathway. Analogous results were reported by Säälik et al
. for CPP/avidin complexes (103
). However, it should be kept in mind, that the interpretation of these particular experimental data is problematic, as many aspects of endocytosis are still poorly understood to date. Even more important, the compounds may have additional unknown effects, which could lead to unexpected changes in reporter gene activity and/or affect the RNAi machinery. Besides, the involvement of several pathways of endocytosis could also be a direct result of an inhomogeneous size distribution of the MPGα/siRNA complexes (S.V. and A.T., unpublished data), since for each process only a certain range of particle sizes is believed to be predominantly taken up (50
). Thus, the size of the complexes may determine their uptake route and consequently their fate. Then again, we cannot entirely rule out that a certain percentage of complexes are taken up via a direct penetration of the plasma membrane as reported by others (39
). In this context it would be very interesting to know the minimal number of siRNA molecules needed to trigger RNAi-mediated half maximal inhibition of luciferase activity. In an attempt to answer this question, we performed technically challenging nuclear microinjection studies of a luciferase expression plasmid together with siR206 into HeLa and ECV304 cells, respectively (). From such experiments it could be calculated that ~300 siRNA molecules per cell were necessary to observe a half maximal inhibition of luciferase activity. Surprisingly, this number is very close to what was determined for LF2000-mediated delivery. Microscopic studies of fluorescently labelled siRNA delivered with LF2000 on the other hand, showed a vesicular distribution similar to what has been observed with MPGα, indicative of the majority of siRNA molecules being not bio-available either (Supplementary Figure 5). Thus, the microinjection approach, at least in our hands, turned out not to be practicable to answer this question. A possible cause could simply be that the siRNA is retained inside the nucleus. Then again, this could not be verified by microinjection of fluorescently labelled siRNA as initially strongly visible fluorescence in the nucleus completely disappeared within 30 min, probably either due to quenching effects or transport into cytoplasm. In spite of this, transiently transfected cells might differ considerably from stably transfected ones.
In conclusion, our data clearly show that a vesicular (presumably endosomal) accumulation of the cargo and not uptake per se is the bottleneck of MPGα-mediated RNA delivery rendering the vast majority of intracellular siRNA cargo molecules inactive. Thus, like for other non-viral delivery systems, solving the problem of vesicular escape remains the main challenge for this CPP approach. Nonetheless, this approach yields IC50 values for siRNA delivery in the sub-nanomolar range far superior to other CPP systems together with the advantage of non-covalent complex formation. Moreover, this study describes a combination of techniques which represent easy to handle tools suitable for a detailed analysis of carrier-mediated delivery of therapeutically interesting nucleic acid molecules, which eventually may lead to considerably improved delivery.